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Method for producing monoclonal antibodies

Inactive Publication Date: 2005-08-18
EURO LAB FUER MOLEKULARBIOLOGIE EMBL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0002] The development of monoclonal antibody-producing cell lines by somatic fusions of B lymphocytes with myeloma cells was first described by Kohler and Milstein over 25 years ago (Kohler & Milstein, 1975). Since then, monoclonal antibodies have played a central role in the exponential growth of our understanding of human physiology, biochemistry and genetics. Monoclonal antibodies are versatile and sensitive tools for detecting and localising specific biological molecules. Monoclonal antibodies can be made against any cell molecule, enabling that molecule to be identified, localised and purified. In some cases, monoclonal antibodies also help identify the function of the molecules to which they bind.
[0075] This method has an advantage over methods disclosed in the prior art in that it enables the simultaneous production of more than one monoclonal antibody, each of which binds to a different purified candidate antigen.

Problems solved by technology

As already discussed, current methods for producing monoclonal antibodies against more than one antigen involve laborious immunisation and isolation protocols for each individual antigen.

Method used

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  • Method for producing monoclonal antibodies
  • Method for producing monoclonal antibodies
  • Method for producing monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

example 1

Immunization with 10 Protein Antigens

Immunization

[0089] An 8-week old female Balb / c mouse was injected intrasplenically with 10 μg of each of 10 protein antigens in 100 μl phosphate buffered saline (PBS). On the third day following (day 3) the mouse was injected intraperitoneally with lug of each of the same 10 protein antigens in 100 μl PBS. On day 5, the same mouse was injected intravenously with 0.1 μg of each of the same 10 protein antigens. On Day 8 the mouse was killed by cervical dislocation and the spleen removed and collected into Dulbecco's Modified Eagle's Medium (DMEM: Life Technologies Inc.).

Fusion

[0090] All steps are performed under sterile or aseptic conditions in a laminar flow hood. The spleen was rendered into a single-cell suspension by mechanical disruption between two frosted-end glass microscope slides. The suspension was filtered into a 50 ml bar-coded conical-bottomed tube (BD Falcon) through a 70 μm nylon cell strainer (BD Falcon) and transferred to th...

example 2

Immunization with Nine Antigens

Immunization:

[0113] A mouse was injected with 25 μg of nine antigens, including 25 μg of a fusion of the antigens B5 and Ket94 / 95, each antigen being mixed with 10 μg CpG DNA and adsorbed onto alum adjuvant (Imject Alum from Pierce). Half of each antigen was administered intraperitoneally and half subcutaneously.

[0114] The mouse was boosted 21 days later with 10)1 g of each antigen mixed with 10)1 g of CpG DNA and adsorbed onto alum adjuvant, half of which was administered intraperitoneally and half subcutaneously.

[0115] Five days after the boost, the spleen was removed.

Fusion and Cell Culture

[0116] The fusions and cell culture were performed as described in Example 1.

Microarray Screening of Antibodies Against B5 and Ket94 / 95.

[0117] An aminosilane glass slides was homogenously coated with purified B5 by dropping 40 μl of ddH2O containing 5 μg of purified B5 and covering with a 22*60 mm coverslip for 60 min at room temperature. The same proce...

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Abstract

The present invention relates to methods for producing monoclonal antibodies. In particular, the invention relates to high throughput methods for producing and screening monoclonal antibodies more rapidly than conventional methods.

Description

[0001] The present invention relates to methods for producing monoclonal antibodies. In particular, the invention relates to high throughput methods for producing monoclonal antibodies more rapidly than conventional methods. [0002] The development of monoclonal antibody-producing cell lines by somatic fusions of B lymphocytes with myeloma cells was first described by Kohler and Milstein over 25 years ago (Kohler & Milstein, 1975). Since then, monoclonal antibodies have played a central role in the exponential growth of our understanding of human physiology, biochemistry and genetics. Monoclonal antibodies are versatile and sensitive tools for detecting and localising specific biological molecules. Monoclonal antibodies can be made against any cell molecule, enabling that molecule to be identified, localised and purified. In some cases, monoclonal antibodies also help identify the function of the molecules to which they bind. [0003] The diagnostic and therapeutic potential of monoclo...

Claims

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Application Information

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IPC IPC(8): C07K16/00C12N5/20C12N15/06
CPCC07K16/00
Inventor SAWYER, ALAN MICHAELDE MASI, FEDERICO
Owner EURO LAB FUER MOLEKULARBIOLOGIE EMBL